This invention relates to the field of protein methylation. More particularly, the present invention relates to antibodies that specifically recognizes peptides and proteins containing methylated arginines.
Protein methylation is considered to play an important role in cellular functions such as signaling. In eukaryotic cells, proteins are methylated on carboxyl groups or on the side chain nitrogen of the amino acids lysine, arginine or histidine.
N-methylation, such as that occurring on arginine residues in proteins, has generally been regarded as a constitutive and irreversible post-translational modification. However, there may be exceptions to this view. For example, NGF has been reported to dramatically alter the pattern of protein methylation observed in PC12 cells after metabolic radiolabeling of protein in intact cells, and by in vitro labeling of proteins in cell extracts.
The enzymes responsible for protein methylation, protein arginine methyl transferases (PRMT) are currently classified into two distinct categories. One type of activity (Type I) produces asymmetric dimethylation of the n1 terminal guanidino nitrogen of arginines in substrate proteins, particularly glycine- and arginine-rich (GAR) segments of proteins. Proteins that are substrates for this reaction include nucleolin, fibrillarin, and several hnRNPs. Type II arginine methyltransferase activity produces symmetric dimethylation of both terminal nitrogens of specific protein arginines. Myelin basic protein is a recognized substrate for this activity.
While protein methylation is recognized to be important in cell signaling mechanisms, its precise role in neuronal growth and differentiation remains to be elucidated. Biochemical analysis of cellular protein methylation is currently hindered by the lack of a simple means of determining the methylation status of native cellular proteins. Mass spectroscopy, though precise, is a very specialized and time-consuming technique. Metabolic radiolabeling of methyl-proteins is problematic owing to the vicissitudes of a variety of kinetic parameters related to cellular enzymes and protein substrates. Thus, there is an ongoing need to develop tools that will enable detection of methylarginine proteins, detection of their methylation status and detections of compositions that affect methylation.
The present invention provides antibodies to arginine methylated proteins. By using these antibodies, the methylation status of cellular proteins involved in cell metabolism and function can be characterized.
The present invention also provides a method of producing antibodies that discriminates between arginine methylated and non-arginine-methylated proteins or peptides. These antibodies are prepared by using peptides containing one or more methylated arginines. The antibodies are reactive against the methylated form of the peptides.
The present invention also provides a method for detecting the presence of methylated proteins in a sample. The method comprises the steps of contacting the sample with antibodies of the present invention and detecting the proteins or peptides bound to the antibody. The method can also be used for identification of compositions that affect methylation of proteins.